Novel insight into molecular mechanism of endotoxin shock: biochemical analysis of LPS receptor signaling in a cell-free system targeting NF-kappaB and regulation of cytokine production/action through beta2 integrin in vivo.

Lipopolysaccharide (LPS), a constituent of gram-negative bacteria cell wall, plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as cytokines, nitrous oxide, superoxide anions, and lipid mediators. In vitro, LPS induces the transcription of a set of genes involved in inflammatory reactions by activating several types of transcription factors, particularly nuclear factor-kappaB (NF-kappaB). An analysis of NF-kappaB activation using a cell-free system demonstated that two pathways converge to activate NF-kappaB; one is staurosporine-sensitive, the other is staurosporine-insensitive and tyrosine kinase-dependent. Furthermore, the latter pathway culminates in IkappaBalpha phosphorylation at serine/threonine residues in its carboxyl-terminal acidic region with dissociation of IkappaBalpha from NF-kappaB, thereby activating NF-kappaB. The requirement for the phosphorylation at this site was confirmed by the specific inhibition of NF-kappaB activation in a cell-free system by the synthetic peptide corresponding to this site. The in vivo administration of an anti-CD18 antibody prevented elevation of plasma tumor necrosis factor (TNF) levels and acute lethality induced by injection of a low dose of LPS into Propionibacterium acnes-primed rabbits or by the administration of a single high dose of LPS into animals. Anti-CD18 also prevented acute lethality induced by one of the main mediators of endotoxin shock, TNF-alpha. Furthermore, an antibody to a ligand for CD18, intercellular adhesion molecule-1, also prevented TNF-induced shock as well as endotoxin shock in rabbits. These observations suggest that the interaction between leukoytes and endothelium through beta2- integrin adhesion molecules may be of primary importance in mediating LPS signals in vivo.